Periodontitis, an inflammatory disease of tissues in the subgingival crevice, can lead to soft tissue damage and dramatic bone loss. Development of periodontitis is associated with a dramatic shift in the subgingival microflora; gram negative microorganisms replace gram positive bacteria as the primary flora. In order to understand why certain bacteria are successful periodontopathogens, it is important to determine how their virulence determinants might be regulated by components of the complex and unique subgingival microenvironment. This proposal focuses on regulation in the gram negative, coccobacillus Actinobacillus actinomycetemcomitans (Aa) for several reasons: (1) Aa is the primary candidate pathogen in several forms of periodontitis, (2) Aa is the only periodontopathogen to produce a leukotoxin, and (3) the Aa leukotoxin gene has been cloned, so both molecular genetic and biochemical approaches can be used to study its regulation. Our hypothesis is that changes in environmental cues will alter the expression of Aa leukotoxin. Aa strains in which both leukotoxin and beta-galactosidase are transcribed from the leukotoxin promoter have been generated. This will allow leukotoxin promoter activity to be assessed using a simple, quantitative beta-galactosidase assay. These Aa strains will be grown in media with varying amounts of iron, in media at different pHs and in cultures which have been stressed (heat shocked). Beta-galactosidase activity will be assayed to determine those culture changes that modulate leukotoxin expression. then RNA and protein will be isolated from the same cultures and analyzed using specific DNA and antibody probes to determine the level at which leukotoxin regulation occurs. the regulation of cell envelope proteins will also be examined from the same cells. The results will show how the virulence factor, leukotoxin, is regulated by changes in these three environmental signals and will also identify other regulated proteins. the other regulated proteins are candidate virulence factors. Using the cloned leukotoxin gene as a starting point, molecular genetic approaches will then be used to determine the DNA sequences and regulatory proteins involved in regulating leukotoxin transcription in Aa. Deletion and point mutant analyses of the leukotoxin promoter will define important cis elements. Transposon mutagenesis will be used to identify genes which regulate the transcriptional response of the leukotoxin gene. The results from these studies will define the cast of characters used by Aa to regulate a virulence factor, leukotoxin. this will enable us, in future years, to delineate the molecular mechanisms by which environmental cues regulate leukotoxin and other virulence factors of Aa.